Project Summary The long-term goal of this R21 proposal is to introduce the NanoVelcro vimentin(+)-CTC Assay as a novel clinical test in hepatocellular carcinoma (HCC) for the detection of underlying aggressive or occult metastatic disease to aid in the selection of HCC candidates for liver transplantation (LT). Currently, LT is the only curative therapy for HCC patients with unresectable, non-metastatic disease, with selection of candidates based solely on radiologic staging systems incorporating tumor size and number (e.g. Milan criteria). Despite these strict criteria, 15-20% of eligible HCC patients experience tumor progression and dropout during the waitlist period, and up to 20% of patients receiving LT develop HCC recurrence ? a major cause of allograft loss and patient mortality. There remains an unmet need to balance this risk of wait-list dropout while minimizing post-LT recurrence when prioritizing HCC candidates for LT. Circulating tumor cells (CTCs) are rare cancer cells found in the bloodstream. NanoVelcro assay, a novel cellular isolation technology introduced by Prof. Tseng (co-I), can identify CTCs in small volumes of blood. Using the NanoVelcro assay, we have identified an HCC CTC phenotype with positive vimentin expression (vimentin(+)-CTCs), that are associated with advanced stage HCC patients who are ineligible for LT. The potential for CTCs to serve as a biomarker of aggressive and/or occult metastatic HCC holds great promise to augment the ability of current radiologic criteria to select HCC patients for LT. Vimentin overexpression in primary HCC has been linked to more aggressive tumor biology, inferior survival outcomes, and the establishment of tumor initiating capacity critical for metastases. In our preliminary studies, vimentin(+) CTCs discriminated early and advanced stage disease with high accuracy, and independently predicted overall and progression-free survival. We hypothesize that detection of vimentin(+)-CTCs reflects an underlying progressive/metastatic disease state, and hence would identify HCC candidates at high-risk for wait-list dropout and post-LT recurrence. This hypothesis will be tested by 1) validating the NanoVelcro HCC CTC assay by comparing fidelity of enumeration data between matched fresh and banked samples, as well as comparison to the FDA approved CellSearchTM Assay and serum AFP (aim 1); and 2) analyzing CTC samples from HCC patients with known waitlist and post-LT outcomes that have been collected and annotated over the past 3 years in our UCLA Liver Biobank (aim 2)? a unique and invaluable resource. We anticipate that the NanoVeclro CTC Assay will efficiently capture HCC CTCs and vimentin(+)-CTCs, and that the vimentin(+)-CTCs will more accurately discriminate HCC patients with/without wait-list dropout and post-LT recurrence compared to the standard radiological staging systems. The successful development of the proposed NanoVelcro vimentin(+)-CTC Assay is rapidly translatable, leading to a powerful predictive assay to identify aggressive and/or occult metastatic HCC not suitable for LT.